[Innovative use of the vascular endothelial growth factor in an experimental model of acute liver failure]. / Utilizzo innovativo del fattore di permeabilità vascolare in un modello sperimentale di insufficienza epatica acuta.

Vascular Endothelial Growth Factor (VEGF) is an endothelial cell mitogen and an important stimulator of sinusoidal endothelial cell proliferation. The aim of this research was to study the effects of exogenous VEGF in a rat model of acute liver failure. The study was conducted on 64 rats (240-300 g). All rats underwent intraperitoneal injection (5 ml/kg) of 25% carbon tetrachloride (CCl4) and 75% paraffin oil. This dosage of CCl4 was devised to induce nonfatal acute liver failure with spontaneous recovery in 7 days. The animals were randomly divided into 2 groups. Group B animals underwent i.v. injection of 200 ng of VEGF165 one hour following intra-peritoneal injection of CCl4. To obtain daily liver functional tests (LFTS) and histological liver samples, 4 rats in each group were sacrificed daily up to 8 days. In group A, the liver histology showed massive periportal hepatocyte necrosis associated with portal lymphocytic infiltrates. The peak of the damage was documented at 72 hours following CCl4. Group B showed minimal necrosis, moderate periportal edema and a minimum periportal steatosis. At 48 hours steatotic changes had disappeared and the periportal edema was resolving. LFTs demonstrated severe liver damage in rats in group A. In group A the peak AST (mean 322.5 IU/L) and ALT (mean 250.25 IU/L) were recorded at 72 hours. In group B, at 72 hours the mean AST was 137 IU/L (normal < 95 IU/L) and ALT 68 IU/L(normal < 45 IU/L). The maximum levels of AST and ALT, in group B, were 152.3 IU/L and 72.3 IU/L, at 24 hours. According to our results exogenous VEGF successfully protects the liver from CCl4 induced acute liver failure. Further studies will demonstrate if exogenous VEGF can be effective in other liver injuries.

Adenosine receptor agonists: synthesis and biological evaluation of the diastereoisomers of 2-(3-hydroxy-3-phenyl-1-propyn-1-yl)NECA.

Among the recently reported 2-(ar)alkynyl derivatives of 5'-N-ethylcarboxamidoadenosine (NECA), the (R,S)-2-(3-hydroxy-3-phenyl-1-propyn-1-yl)NECA [(R,S)-PHPNECA or SCH 59761] was found to be a very potent agonist at A1 and A2A receptor subtypes, with a Ki of 2.5 nM and 0.9 nM, respectively. Furthermore, this compound showed an inhibitory activity on platelet aggregation 16-fold higher than NECA, being the most potent anti-aggregatory nucleoside reported so far. Since this compound bears a chiral carbon in the side chain, the diastereoisomer separation was undertaken both by chiral HPLC and by a stereospecific synthetic method. Binding assays have shown that the (S)-diastereomer is about fivefold more potent and selective than the (R)-diastereomer as agonist of the A2A receptor subtype [(S)-PHPNECA, KiA2A = 0.5 nM; (R)-PHPNECA, KiA2A = 2.6 nM]. Functional studies indicated that (S)-PHPNECA possesses marked vasodilating activity and produces a relevant decrease in heart rate. Moreover, the (S)-diastereomer proved to be about ten times more potent than the (R)-diastereomer in inducing cardiovascular effects, in in vivo hemodynamic studies. However, the greatest difference between these two enantiomers resulted in the platelet aggregation test: in fact, the (R)-diastereomer displayed an inhibitory activity similar to that of NECA, whereas the (S)-diastereomer was 37-fold more active than NECA as an inhibitor of rabbit platelet aggregation, induced by ADP. These data suggest that (S)-PHPNECA could be a useful tool to investigate the mode of binding of agonists to the platelet adenosine receptor subtype.

2-alkenyl and 2-alkyl derivatives of adenosine and adenosine-5'-N-ethyluronamide: different affinity and selectivity of E- and Z-diastereomers at A2A adenosine receptors.

A series of new 2-(ar)alkenyl, both Z- and E-diastereomers, and 2-alkyl derivatives of adenosine-5'-N-ethyluronamide (NECA) and adenosine were synthesized and evaluated for their interaction with the A1 and A2A adeosine receptors, to better understand the conformational requirements of the receptor area interacting with the substituents in the 2- and 5'-positions. Partial reduction of the triple bond in 2-alkynyl derivatives of NECA led to compounds whose activity at the A2A receptor subtype was related to Z-E-isomerism, the E-diastereomers being more potent and selective than the Z-ones. Saturation of the side chain markedly reduced compound affinity at adenosine receptors. Specifically, compounds bearing an (E)-alkenyl chain, while maintaining the same affinity at A2A receptors as the corresponding alknyl derivatives, showed an increase in A2A vs A1 selectivity. Hence, the new nucleosides (E)-2-hexenylNECA (12a) and (E)-2-(phenylpentenyl)NECA (12b) exhibited both high A2A receptor affinity (Ki = 1.6 and 3.5 nM, respectively) and A2A vs A1 selectivity (157- and 290-fold, respectively). Moreover, 12a displayed potent antiaggregatory activity, similar to that of the reference compound NECA. Comparison between NECA and adenosine derivatives further demonstrated that the 5'-ethylcarboxamido group is critical for the A2A affinity. These studies indicated that the orientation of the substituent in the 2-position and the nature of the 5'-group in adenosine derivatives are critical to achieve high affinity and selectivity at the A2A adenosine receptor subtype.